CN105925626A - Method of extracting polymalic acid - Google Patents
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Abstract
The invention discloses a method of extracting polymalic acid, combining fermenting cultivation of polymalic acid and membrane separation. After fermentation is stable, sampling and membrane filtering are performed, penetrating substances are graded and extracted, high-molecular-weight substances and bacteria intercepted flow back, and polymalic acid polymerization is continued; traditional independent fermentation and extraction processes are abandoned, operating efficiency is improved, extraction process cost is lowered, different molecular weights of polymalic acid may be extracted at a time by selecting ultrafiltration membranes with different intercept molecular weights, and this method is an effective method to prepare efficiently different molecular weights of polymalic acid.
Description
Technical field
The present invention relates to high molecular polymer preparation field, particularly relate to the extraction side of a kind of polymalic acid
Method.
Background technology
Aureobasidium pullulans have another name called sprout grow sturdily mould, classification belong to deuteromycetes, be that one has yeast type and bacterium
The multiform fungus of wire type form.Aureobasidium pullulans can synthesize during the fermentation polymalic acid, antibiotics,
The multi-products such as enzyme, extracellular polysaccharides and single cell protein.It is reported that the utilization such as LIUSJ is sprouted short
Obstruct mould fermentation and obtained polymalic acid, and find that the generation of polymalic acid is in logarithmic (log) phase.
Polymalic acid is the high molecular polymer synthesized for only monomer with malic acid, unique chemical constitution
Make it have the advantageous properties such as biocompatibility, biodegradability and Bioabsorbable.Next, it
As a kind of water solublity aliphatic polyester, there is high water soluble, the most derivative.Polymalic acid
Unique character so that it is have in terms of the many such as medicine, cosmetics, environmental improvement and essence and flavoring agent
Wide application prospect.Biological synthesis process synthesis polymalic acid is mainly synthesized in fermentation by microorganism
After, it is secreted into outside born of the same parents, is present in fermentation liquid, more i.e. can obtain poly-Fructus Mali pumilae through follow-up extraction process
Acid product, compares with chemical synthesis, and bioanalysis has low cost, reaction condition gentleness, molecular weight phase
To relatively advantages of higher, therefore bioanalysis synthesis polymalic acid has vast potential for future development.
Polymalic acid is applicable to pharmaceutical carrier field, and its molecular weight is to evaluate it as pharmaceutical carrier
The important indicator of energy, it is possible to the polymer molecule weight range as pharmaceutical carrier is 1.5～200kDa, at this
In the range of, properly increase polymalic acid molecular weight, its drug loading can be improved, and when molecular weight is too high,
Then it is difficult to lose the performance as pharmaceutical carrier through cell membrane.
At present, the method for relevant fermentable synthesis polymalic acid has multiple, including mutagenic obtained
Superior strain, optimization of fermentation conditions and culture medium improve yield, the compound somatomedin that adds improves yield
Etc., also have about the patent in terms of polymalic acid extraction, including organic solvent extraction, Quan Shui
Changing extraction etc., laboratory once used and changes fermentation condition or add the method life of a certain somatomedin
Producing the polymalic acid of different molecular weight, the method can only be for the poly-Fructus Mali pumilae synthesizing a certain specified molecular weight
Acid.
Summary of the invention
In view of this, it is contemplated that propose the extracting method of a kind of polymalic acid, to solve prior art
Middle polymalic acid molecular weight is big, and once extracts the problem that polymalic acid molecular weight is single.
For reaching above-mentioned purpose, the technical scheme is that and be achieved in that: the carrying of a kind of polymalic acid
Access method, including seed culture, fermentation culture, separation and Extraction and the mensuration of polymalic acid purity, wherein
The method of separation and Extraction is as follows:
(1) when seed liquor fermentation culture 48-60h, start to be separated by fermentation liquid, at fermentation tank
Discharge outlet is equipped with ultrafilter membrane, takes out 300～500ml every 8-12h from fermentation tank discharge outlet membrane filtration
Fermentation liquid permeate, thalline and macromolecular substances are trapped in fermentation tank continuation reaction, the most past
Fermentation tank adds and the fermentation medium of permeate same volume, it is thus achieved that fermentation liquid sequentially pass through and retain
Molecular weight is that the ultrafilter membrane of 20KDa, 10KDa, 5KDa carries out ultrafiltration；
(2) by step (1) sequentially passes through three kinds of filtrates that three kinds of ultrafilter membrane ultrafiltration obtain, enter respectively
Row activated carbon decolorizing, carries out vacuum lyophilization after then concentrating respectively through rotary evaporation, it is thus achieved that three kinds
The polymalic acid of molecular weight.
Preferably, the method for seed culture is: by Aureobasidium pullulans slant activation 4-5h, then uses sterilized water
Wash lower spore, prepare spore suspension, be transferred to spore suspension cultivate equipped with in the container of seed culture medium,
Cultivation temperature is 25-35 DEG C, rotating speed 160-200r/min, cultivates 30-60h.
Preferably, the method for fermentation culture is: aseptically, accesses seed liquor equipped with fermentation culture
Cultivating in the fermentation tank of base, condition of culture is rotating speed 500-700r/min, and tank pressure is 0.1-0.3Mpa,
Ventilating ratio 1:6-1:4.
Preferably, the mensuration of polymalic acid purity uses high performance liquid chromatography, and first weighing quality is m1
Polymalic acid, be configured to the solution that concentration is 2g/L, then take 5mL polymalic acid with pipet molten
Liquid adds isopyknic 1mol/L H2SO4Solution, hydrolyzes 10h, by poly-Fructus Mali pumilae under the conditions of 110 DEG C
Acid complete hydrolysis is monomer malic acid；Then with the containing of malic acid before and after Water By High Performance Liquid solution
Amount, the standard curve in conjunction with malic acid draws the content of malic acid in polymalic acid, and before hydrolysis, malic acid contains
The content m2 that difference is polymalic acid of the content of malic acid after amount and hydrolysis, the purity of polymalic acid is:
Preferably, consisting of of seed culture medium: sucrose 80-120g/L, yeast extract 2-4g/L, ammonium sulfate
1-3g/L, succinic acid 3-5g/L, KH2PO40.2-0.8g/L, MgSO4·7H2O 0.05-0.15g/L,
ZnSO4·7H2O 0.03-0.06g/L, remaining is distilled water.
Preferably, consisting of of fermentation medium: sucrose 100-300g/L, peptone 20-40g/L,
KH2PO40.05-0.13g/L, NaNO33-5g/L, MgSO4·7H2O 0.3-0.9g/L, KCl
0.2-0.8g/L, MnSO40.01-0.08g/L, remaining is distilled water.
Preferably, consisting of of seed culture medium: sucrose 100g/L, yeast extract 3g/L, ammonium sulfate 2g/L,
Succinic acid 4g/L, KH2PO40.5g/L, MgSO4·7H2O 0.1g/L, ZnSO4·7H2O 0.05g/L,
Remaining is distilled water.
Preferably, consisting of of fermentation medium: sucrose 200g/L, peptone 30g/L, KH2PO4
0.1g/L, NaNO34g/L, MgSO4·7H2O 0.6g/L, KCl 0.5g/L, MnSO40.05g/L,
Remaining is distilled water.
Relative to prior art, a kind of polymalic acid extracting method of the present invention, have the advantage that
(1) extracting method of a kind of polymalic acid of the present invention, by fermentation culture polymalic acid and
Membrane separation technique combines cleverly, and after fermentation enters stable phase, sampling carries out membrane filtration, passes through
Material grading extraction, the material of the macromolecule simultaneously retained and thalline backflow, continue synthesize poly-Herba Marsileae Quadrifoliae
Fruit acid, has abandoned traditional independence fermentation and extraction process, has improve work efficiency, reduce extraction work
The cost of skill.
(2) extracting method of a kind of polymalic acid of the present invention, can retain according to selecting difference
The ultrafilter membrane of molecular weight disposably extracts the polymalic acid of different molecular weight, is a kind of efficiently preparation difference point
The effective ways of son amount polymalic acid.
Detailed description of the invention
Below in conjunction with specific embodiment, present disclosure is further illustrated.
Case study on implementation 1:
(1) seed culture
By Aureobasidium pullulans slant activation 4h, then with spore under aseptic washing, prepare spore suspension, will
Spore suspension is transferred in the container containing seed culture medium cultivate, cultivation temperature 25 DEG C, rotating speed 160r/min,
Cultivating 30h, wherein seed culture medium consists of: sucrose 80g/L, yeast extract 2g/L, ammonium sulfate 1g/L,
Succinic acid 3g/L, KH2PO40.2g/L, MgSO4·7H2O 0.05g/L, ZnSO4·7H2O 0.03g/L,
Remaining is distilled water；
(2) fermentation culture
Aseptically, the seed liquor obtained in step (1) is accessed sending out equipped with fermentation medium
Cultivating in ferment tank, condition of culture is rotating speed 500r/min, tank pressure 0.1Mpa, ventilating ratio 1:4, its
Middle fermentation medium is: sucrose 100g/L, peptone 20g/L, KH2PO40.05g/L, NaNO33g/L,
MgSO4·7H2O 0.3g/L, KCl 0.2g/L, MnSO40.01g/L, remaining is distilled water；
(3) separation of fermentative broth, filtration
When seed liquor fermentation culture 48h in step (2), start to separate fermentation liquid, sending out
Ferment tank discharge outlet is provided with ultrafilter membrane, takes out 300ml every 8h from fermentation tank discharge outlet membrane filtration
Fermentation liquid permeate, retains thalline and macromolecular substances returns to continue in fermentation tank reaction, the most past
In fermentation tank add 300ml fermentation medium, it is thus achieved that fermentation liquid with molecular cut off be successively
The ultrafilter membrane of 20KDa, 10KDa, 5KDa carries out ultrafiltration；
(4) preparation of polymalic acid
Filtrate between the three kinds of molecular weight areas obtain ultrafiltration in step (3), carries out activated carbon respectively and takes off
Color, carries out vacuum lyophilization after then concentrating respectively through rotary evaporation；
(5) mensuration of polymalic acid purity
High performance liquid chromatography is used to carry out the polymalic acid of the three kinds of molecular weight obtained in step (4)
The assay of polymalic acid, the purity recording polymalic acid all reaches more than 80.44%.
Case study on implementation 2:
(1) seed culture
By Aureobasidium pullulans slant activation 5h, then with spore under aseptic washing, prepare spore suspension, will
Spore suspension is transferred in the container containing seed culture medium cultivate, cultivation temperature 35 DEG C, rotating speed
200r/min, cultivates 60h, and wherein seed culture medium consists of: sucrose 120g/L, yeast extract 4g/L,
Ammonium sulfate 3g/L, succinic acid 5g/L, KH2PO40.8g/L, MgSO4·7H2O 0.15g/L,
ZnSO4·7H2O 0.06g/L, remaining is distilled water；
(2) fermentation culture
Aseptically, the seed liquor obtained in step (1) is accessed sending out equipped with fermentation medium
Cultivating in ferment tank, condition of culture is rotating speed 700r/min, tank pressure 0.3Mpa, ventilating ratio 1:6, its
Middle fermentation medium is: sucrose 300g/L, peptone 40g/L, KH2PO40.13g/L, NaNO35g/L,
MgSO4·7H2O 0.9g/L, KCl 0.8g/L, MnSO40.08g/L, remaining is distilled water；
(3) separation of fermentative broth, filtration
When seed liquor fermentation culture 54h in step (2), start to separate fermentation liquid, sending out
Ferment tank discharge outlet is provided with ultrafilter membrane, takes out 400ml every 10h from fermentation tank discharge outlet membrane filtration
Fermentation liquid permeate, retains thalline and macromolecular substances returns to continue in fermentation tank reaction, the most past
In fermentation tank add 400ml fermentation medium, it is thus achieved that fermentation liquid with molecular cut off be successively
The ultrafilter membrane of 20KDa, 10KDa, 5KDa carries out ultrafiltration；
(4) preparation of polymalic acid
Filtrate between the three kinds of molecular weight areas obtain ultrafiltration in step (3), carries out activated carbon respectively and takes off
Color, is then passed through carrying out vacuum lyophilization after rotary evaporation concentrates；
(5) mensuration of polymalic acid purity
High performance liquid chromatography is used to carry out the polymalic acid of the three kinds of molecular weight obtained in step (4)
The assay of polymalic acid, the purity recording polymalic acid all reaches more than 84.03%.
Case study on implementation 3:
(1) seed culture
By Aureobasidium pullulans slant activation 4.5h, then with spore under aseptic washing, prepare spore suspension,
It is transferred to spore suspension in the container containing seed culture medium cultivate, cultivation temperature 28 DEG C, rotating speed
180r/min, cultivates 40h, and wherein seed culture medium consists of: sucrose 100g/L, yeast extract 3g/L,
Ammonium sulfate 2g/L, succinic acid 4g/L, KH2PO40.5g/L, MgSO4·7H2O 0.1g/L,
ZnSO4·7H2O 0.05g/L, remaining is distilled water；
(2) fermentation culture
Aseptically, the seed liquor obtained in step (1) is accessed sending out equipped with fermentation medium
Cultivating in ferment tank, condition of culture is rotating speed 600r/min, tank pressure 0.2Mpa, ventilating ratio 1:5, its
Middle fermentation medium is: sucrose 200g/L, peptone 30g/L, KH2PO40.1g/L, NaNO34g/L,
MgSO4·7H2O 0.6g/L, KCl 0.5g/L, MnSO40.05g/L, remaining is distilled water；
(3) separation of fermentative broth, filtration
When seed liquor fermentation culture 60h in step (2), start to separate fermentation liquid, sending out
Ferment tank discharge outlet is provided with ultrafilter membrane, takes out 500ml every 12h from fermentation tank discharge outlet membrane filtration
Fermentation liquid permeate, retains thalline and macromolecular substances returns to continue in fermentation tank reaction, the most past
In fermentation tank add 500ml fermentation medium, it is thus achieved that fermentation liquid with molecular cut off be successively
The ultrafilter membrane of 20KDa, 10KDa, 5KDa carries out ultrafiltration；
(4) preparation of polymalic acid
Filtrate between the three kinds of molecular weight areas obtain ultrafiltration in step (3), carries out activated carbon respectively and takes off
Color, is then passed through carrying out vacuum lyophilization after rotary evaporation concentrates；
(5) mensuration of polymalic acid purity
High performance liquid chromatography is used to carry out the polymalic acid of the three kinds of molecular weight obtained in step (4)
The assay of polymalic acid, the purity recording polymalic acid all reaches more than 83.95%.
Case study on implementation 4:
(1) seed culture
By Aureobasidium pullulans slant activation 4h, then with spore under aseptic washing, prepare spore suspension, will
Spore suspension is transferred in the container containing seed culture medium cultivate, cultivation temperature 28 DEG C, rotating speed
180r/min, cultivates 40h, and wherein seed culture medium consists of: sucrose 100g/L, yeast extract 3g/L,
Ammonium sulfate 2g/L, succinic acid 4g/L, KH2PO40.5g/L, MgSO4·7H2O 0.1g/L,
ZnSO4·7H2O 0.05g/L, remaining is distilled water；
(2) fermentation culture
Aseptically, the seed liquor obtained in step (1) is accessed sending out equipped with fermentation medium
Cultivating in ferment tank, condition of culture is rotating speed 600r/min, tank pressure 0.2Mpa, ventilating ratio 1:5, its
Middle fermentation medium is: sucrose 200g/L, peptone 30g/L, KH2PO40.1g/L, NaNO34g/L,
MgSO4·7H2O 0.6g/L, KCl 0.5g/L, MnSO40.05g/L, remaining is distilled water；
(3) separation of fermentative broth, filtration
When seed liquor fermentation culture 60h in step (2), start to separate fermentation liquid, sending out
Ferment tank discharge outlet is provided with ultrafilter membrane, takes out 400ml every 10h from fermentation tank discharge outlet membrane filtration
Fermentation liquid permeate, retains thalline and macromolecular substances returns to continue in fermentation tank reaction, the most past
In fermentation tank add 400ml fermentation medium, it is thus achieved that fermentation liquid with molecular cut off be successively
The ultrafilter membrane of 20KDa, 10KDa, 5KDa carries out ultrafiltration；
(4) preparation of polymalic acid
Filtrate between the three kinds of molecular weight areas obtain ultrafiltration in step (3), carries out activated carbon respectively and takes off
Color, is then passed through carrying out vacuum lyophilization after rotary evaporation concentrates；
(5) mensuration of polymalic acid purity
High performance liquid chromatography is used to carry out the polymalic acid of the three kinds of molecular weight obtained in step (4)
The assay of polymalic acid, the purity recording polymalic acid all reaches more than 85.12%.
Claims (8)
1. an extracting method for polymalic acid, including seed culture, fermentation culture, separation and Extraction and
The mensuration of polymalic acid purity, it is characterised in that the method for separation and Extraction is as follows:
(1) when seed liquor fermentation culture 48-60h, start to be separated by fermentation liquid, at fermentation tank
Discharge outlet is equipped with ultrafilter membrane, takes out 300～500ml every 8-12h from fermentation tank discharge outlet membrane filtration
Fermentation liquid permeate, thalline and macromolecular substances are trapped in fermentation tank continuation reaction, the most past
Fermentation tank adds and the fermentation medium of permeate same volume, it is thus achieved that fermentation liquid sequentially pass through and retain
Molecular weight is that the ultrafilter membrane of 20KDa, 10KDa, 5KDa carries out ultrafiltration；
(2) by step (1) sequentially passes through three kinds of filtrates that three kinds of ultrafilter membrane ultrafiltration obtain, enter respectively
Row activated carbon decolorizing, carries out vacuum lyophilization after then concentrating respectively through rotary evaporation, it is thus achieved that three kinds
The polymalic acid of molecular weight.
The extracting method of a kind of polymalic acid the most according to claim 1, it is characterised in that described
The method of seed culture is: by Aureobasidium pullulans slant activation 4-5h, then with spore under aseptic washing,
Prepare spore suspension, be transferred to spore suspension cultivate equipped with in the container of seed culture medium, cultivation temperature
For 25-35 DEG C, rotating speed 160-200r/min, cultivates 30-60h.
The extracting method of a kind of polymalic acid the most according to claim 1, it is characterised in that described
The method of fermentation culture is: aseptically, and seed liquor is accessed the fermentation tank equipped with fermentation medium
In cultivate, condition of culture is rotating speed 500-700r/min, and tank pressure is 0.1-0.3Mpa, ventilating ratio
1:6-1:4。
The extracting method of a kind of polymalic acid the most according to claim 1, it is characterised in that described
The mensuration of polymalic acid purity uses high performance liquid chromatography, first weighs the polymalic acid that quality is m1,
It is configured to the solution that concentration is 2g/L, then takes 5mL polymalic acid solution with pipet and add equal-volume
1mol/L H2SO4Solution, hydrolyzes 10h under the conditions of 110 DEG C, by polymalic acid complete hydrolysis is
Monomer malic acid；Then with the content of malic acid before and after Water By High Performance Liquid solution, in conjunction with Fructus Mali pumilae
The standard curve of acid draws the content of malic acid in polymalic acid, Herba Marsileae Quadrifoliae after the front malic acid content of hydrolysis and hydrolysis
The difference of the content of fruit acid is the content m2 of polymalic acid, and the purity of polymalic acid is:
The extracting method of a kind of polymalic acid the most according to claim 2, it is characterised in that described
Consisting of of seed culture medium: sucrose 80-120g/L, yeast extract 2-4g/L, ammonium sulfate 1-3g/L, fourth
Diacid 3-5g/L, KH2PO40.2-0.8g/L, MgSO4·7H2O 0.05-0.15g/L, ZnSO4·7H2O
0.03-0.06g/L, remaining is distilled water.
The extracting method of a kind of polymalic acid the most according to claim 3, it is characterised in that described
Consisting of of fermentation medium: sucrose 100-300g/L, peptone 20-40g/L, KH2PO4
0.05-0.13g/L, NaNO33-5g/L, MgSO4·7H2O 0.3-0.9g/L, KCl 0.2-0.8g/L,
MnSO40.01-0.08g/L, remaining is distilled water.
The extracting method of a kind of polymalic acid the most according to claim 5, it is characterised in that described
Consisting of of seed culture medium: sucrose 100g/L, yeast extract 3g/L, ammonium sulfate 2g/L, succinic acid
4g/L, KH2PO40.5g/L, MgSO4·7H2O 0.1g/L, ZnSO4·7H2O 0.05g/L, remaining is
Distilled water.
The extracting method of a kind of polymalic acid the most according to claim 6, it is characterised in that described
Consisting of of fermentation medium: sucrose 200g/L, peptone 30g/L, KH2PO40.1g/L, NaNO3
4g/L, MgSO4·7H2O 0.6g/L, KCl 0.5g/L, MnSO40.05g/L, remaining is distilled water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148436A (en) * | 2016-09-18 | 2016-11-23 | 天津北洋百川生物技术有限公司 | A kind of extracting method preparing different molecular weight polymalic acid |
| CN107723318A (en) * | 2017-11-28 | 2018-02-23 | 申国庆 | The high yield culture medium of polymalic acid |
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| CN105586369A (en) * | 2015-12-15 | 2016-05-18 | 天津北洋百川生物技术有限公司 | Method for preparing polymalic acid by adding oxygen carriers |
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2016
- 2016-06-07 CN CN201610409712.8A patent/CN105925626B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105586369A (en) * | 2015-12-15 | 2016-05-18 | 天津北洋百川生物技术有限公司 | Method for preparing polymalic acid by adding oxygen carriers |
Non-Patent Citations (2)
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| 刘森泉等: "反相高效液相色谱法测定发酵液中β-聚苹果酸含量", 《食品与发酵工业》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148436A (en) * | 2016-09-18 | 2016-11-23 | 天津北洋百川生物技术有限公司 | A kind of extracting method preparing different molecular weight polymalic acid |
| CN107723318A (en) * | 2017-11-28 | 2018-02-23 | 申国庆 | The high yield culture medium of polymalic acid |
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